1. Field of the Invention
This invention relates generally to apparatus and methods for differentiating constituent types of formed bodies (e.g., cells or other small particles) in liquid samples (e.g., whole blood or other particle-suspending liquids). More particularly, this invention relates to improvements in optical flow cells of the type commonly used in flow cytometers (e.g., hematology and fluorescence flow cytometer instruments) that function to sense, differentiate, and characterize formed bodies by various optical transduction means, preferably in combination with non-optical parameters acquired via the Coulter Principle. The invention further relates to improvements in methods for making optical flow cells, by which a flow channel of polygonal cross-section is provided within a monolithic transparent element.
2. Discussion of the Prior Art
It is common practice to automate analyses of patient body fluids as an aid in diagnosing a patient's state of health. Such analyses typically include flowing a prepared portion of such body fluids through a transducer to derive certain parameters characteristic of the several different types or subpopulations of constituent formed bodies therein, differentiating and enumerating the several types or subpopulations of the formed bodies on the basis of the derived parameters, and processing or correlating the resultant information to provide desired diagnostics. For example, these tasks can be accomplished for whole blood via characterization of blood cells therein by conventional automated hematology analyzers and flow cytometers.
The fundamental performance limit of instruments of the type noted above originates in the transduction of formed-body properties into the characterizing parameters used to assign individual formed bodies to specific subpopulations. For many applications, optical transductive methods alone provide effective means for characterizing formed bodies. In these applications, a portion of a prepared sample is interrogated with optical radiation as it flows through a channel formed in an optically transparent element, or flow cell, forming a portion of an opto-electric transducer. Suitable photo-detectors, also forming part of the transducer, are positioned to detect various optical parameters from an irradiated formed body, including, for example, its optical absorbance of the interrogating beam, its fluorescence at different wavelengths, and its light-scattering effect within one or more angular ranges. In these optical-only applications, it will be appreciated that the physical extent of the flow-cell channel can be relatively large without adversely affecting the determination of these optical parameters. However, in other cytometric applications where such optical parameters are combined with simultaneously determined non-optical parameters, in particular those based on the Coulter Principle (discussed below), the transverse cross-section and length of the flow-cell channel must be dramatically restricted to achieve suitable signal strength.
Due to the low sensitivity of then-existing optical sensing methods, W. H. Coulter devised an electronic method for characterizing minute formed bodies suspended in a liquid. The now well-known Coulter Principle enables determination of the volume of formed bodies, by flowing a sample portion prepared in an electrically conductive liquid through a particle-sensing (or volumeter) conduit simultaneously with an electric current. The electrical resistivity of the particle-suspending liquid differs from that of the particles, the electrical contrast permitting counting and sizing of particles transiting the volumeter conduit. Although other geometries were discussed in Coulter's U.S. Pat. No. 2,656,508, volumeter conduits are usually cylindrical bores in a thin insulative wafer, the conduit's cross-sectional area and length determining volumetric sensitivity, coincidence volume, and maximum passable formed-body dimension; thus, conduit diameters are typically at most an order of magnitude greater than the diameter of the typical formed bodies to be analyzed. The volumeter conduit forms the only fluidic communication between two insulative chambers, with no requirement on the optical characteristics of the wafer material surrounding the conduit. Typically, a direct current (DC) is provided through the conduit, and resistive Coulter volume (V) signals are acquired via a pair of electrodes positioned outside the opposite ends of the volumeter conduit. However, in U.S. Pat. No. 3,502,974 to W. H. Coulter and W. R. Hogg, an excitation current including at least one alternating current (AC) was provided through the conduit, thereby permitting determination of not only the resistive but also reactive components of the conduit current resulting from its modulation by passage of a formed body. When such currents include one having a frequency in the radio-frequency (RF) range (e.g., 23 MHz), the respective components permit estimation of the volume (V) and electrical conductivity (C) of a formed body, and the ratio of the reactive to resistive components is said to be the “opacity” of the formed body.
In commonly assigned U.S. Pat. No. 6,228,652 (hereinafter, the '652 patent), apparatus is disclosed that can provide simultaneous acquisition of various optical, Coulter V, and Coulter C signals from an individual formed body, with subsequent differentiation of formed-body subpopulations in whole blood based thereon. In the '652 patent the preferred flow-cell comprises an optically transparent element having a prismatic exterior envelope of square transverse cross-section, measuring about 4.2 mm on each side, and having a length of about 6.3 mm. (As used herein, the word “prismatic” refers to any three-dimensional figure composed of three or more intersecting sides that are planar, and a pair of opposing ends that are polygonal in shape. “Polygonal” is used herein to refer to any closed plane figure having at least three substantially straight sides, and “planar” as used herein refers to a surface having an area that is predominantly flat.) Centrally located within such prismatic element is a prismatic volumeter conduit having a square transverse cross-section about 50 micra on each side and a length of about 65 micra; the relatively small transverse cross-section and length of the conduit are necessary to attain a reasonable volumetric sensitivity and coincidence volume for acquiring said V and C signals. Thus, the ratio of the respective transverse cross-sectional areas of said conduit and envelope is approximately 0.00014, and the wall thickness is about 2.075 mm. To acceptably limit aberrational content of optical signals, surfaces of the prismatic envelope and conduit must be substantially parallel, with optical planarity. This combination of square/square cross-sectional geometries, wall thickness, wall surface parallelism, and wall flatness is difficult to achieve.
To manufacture prismatic flow cells of the type described in the '652 patent, a relatively complex planarization process has been used wherein four transparent plates, e.g., a form of silica (SiO2, commonly called quartz), are polished to predetermined thickness and finish and assembled as shown in FIG. 13. During assembly, a pair of said plates CC1 and CC3 is spaced apart by the other pair CC2 and CC4 to form two walls of flow cell 30, with the pair of equal-thickness spacer plates appropriately spaced apart a predetermined distance so that their opposing edges complete an internal channel Z. Preferably, these separate complementary elements are of appropriate dimensions and joined at their interfaces by a fusion technique to form a prismatic rod having an internal, longitudinally-extending, straight channel of a desired square transverse cross-section therein. This rod is then cut to a desired length, e.g., 6.3 mm, and polished to the desired external geometry and dimensions to form a flow cell having a prismatic envelope, e.g., opposing sides 50 in FIG. 13 having a flat-to-flat separation of 4.2 mm. Such flow cells having prismatic flow channels of constant longitudinal section but various cross-sections, both square and rectangular, have been made by varying the thickness and separation of the two spacer plates separating the two windows. A prismatic volumeter conduit, e.g., the 50 micra by 50 micra square conduit described above, is formed in such flow cells by boring the square channel from both ends to a suitable diameter (e.g., 1.25 mm), leaving in situ a short length (e.g., 65 micra) of the original channel in the middle of the flow cell that opens at each end into a small cup-shaped recess (e.g., of radius about 600 micra) substantially tangent to a 90-degree cone coaxial with the original channel and continuous with the end bores. The resulting longitudinal channel section is such that the sample liquid in a hydrodynamically-focusing sheath liquid passes centrally through the volumeter conduit within the flow cell. Other embodiments of flow cells made by the planarization process have been adapted to function in fluorescence flow cytometers such as the XL and FC500 research analyzers, as well as the Altra™ cell sorter, all made and sold by Beckman Coulter, Inc. (Single-piece flow cells suited to application in these instruments and made by the method of the present invention are illustrated in respective FIGS. 9, 10, and 11 and will be discussed as embodiments.)
Although useful prismatic flow cells have been produced by the above-described planarization process, the yield of reliably functioning flow cells processed to contain a volumeter conduit has proven to be very low, typically less than 1 in 3. For formed bodies transiting the internal volumeter conduit Z of useful flow cells (e.g., BC2 in FIG. 13), optical signals acquired through the two windows may be substantially repeatable (e.g., the forward scatter (FS) signals resulting from a sensor placed outside window CC1 and on the optical axis OA opposite the entry window CC3 for radiation beam B in FIG. 13). However, those acquired through the two walls comprising spacer plates (e.g., fluorescence (F) signal acquired through CC2 and side-scatter (SS) signal acquired through CC4 in FIG. 13) demonstrate both sensitivity to excitation beam position in individual cases and unit-to-unit variability in the resulting optical signals. More importantly, flow cells made by the planarization process (e.g., flow cell 30 in FIG. 13) are prone to subsequent failure modes: Firstly, irregularities and air pockets in the fused joins characteristic of the planarization approach result in localized heating due to the RF component of the conduit excitation current, with consequent failure of the join; secondly, some flow cells demonstrating longevity under RF exposure show join-dependent unit-to-unit variability in optical signals, even though joins are systematically positioned relative to the axis of optical excitation; thirdly, join imperfections tend to enlarge when sample flows are accompanied by significant cyclic pressure; and finally, flow cells left in stored apparatus tend to separate along the joins due to crystallization of salts if residual reagents are allowed to evaporate.
More-complex production processes, wherein various transparent solids of predetermined geometry are appropriately assembled, have also been used to make optical flow cells having prismatic volumeter conduits of a desired geometry and dimensions. Commonly assigned U.S. Pat. No. 4,348,107 (hereinafter the '107 patent) discloses optical flow cells in which a volumeter conduit having a preferably square transverse cross-section is contained within an envelope having an exterior spherical surface or other surface of revolution. (Single-piece flow cells having this envelope, but made by the method of the present invention, are illustrated in FIGS. 12A, 12B, and 12C and will be discussed as embodiments.) As illustrated in the '107 patent, such flow cells are made by joining together four complementary, truncated, square-based pyramids formed in a transparent material (e.g., quartz). The apex is polished from each pyramid to a depth calculated to yield one side of the desired volumeter conduit, and the pyramids then appropriately assembled and adhesively joined together so that the truncated apexes form an unobstructed square prismatic conduit, the adjacent faces of the joined pyramids forming a tapering longitudinal section at one or both ends of the conduit. Although optical signals may be acquired through the planar walls of the resulting prismatic envelope, it is preferred that means unspecified in the '107 patent then provide the flow cell an envelope formed as a surface of revolution. An extension allowing coupling of the sample liquid and a hydrodynamically-focusing sheath liquid through the conduit is sealed to the resulting flow cell where its surface is intersected by one or both the approaches formed by the exposed sides of the four pyramids. The '107 patent notes that optical and mechanical characteristics of said structure proved suboptimal, the adhesive joins potentially fluorescing or separating, but provides no alternative joining method. A theoretical comparison, of the optical properties of the idealized '107 flow-cell structure with those of a flow cell having a square transverse cross-section in a square prismatic envelope, was published in Applied Optics (26:3244-3248, 1987) by the inventor and one of the present co-inventors; no method for production of either flow-cell structure was described. The inventor and other co-workers later verified some of those theoretical predictions in a second comparison (Cytometry 20:185-190, 1995) using an embodiment of the '107 flow cell having the four pyramids fused together, thus avoiding said adhesive join problems, and a monolithic cylindrical flow cell such as used in automated hematology instruments manufactured and sold by Beckman Coulter, Inc. (discussed below); FIG. 2 in this publication shows the '107 flow cell sealed, after production of a polished spherical exterior envelope, between extensions of the plastic chambers housing electrodes enabling acquisition of Coulter V and C signals. As disclosed in U.S. Pat. Nos. 4,673,288 and 4,818,103, variations of the approach disclosed in the '107 patent have been used to provide prismatic volumeter conduits having a triangular cross-section in a similarly shaped envelope, with square, five-sided, etc., structures said to be within the scope of the invention. To allow efficient collection by a microscope objective of optical signals from such triangular volumeter conduits, in U.S. Patent Application 2007/0085997 a thin transparent plate (window) is substituted for one of the truncated pyramids, with the envelope completed by the remaining two complementary components modified to facilitate interrogation of formed bodies by optical radiation through their walls; this approach is implemented in the Quanta™ cytometer sold by Beckman Coulter, Inc. As will be appreciated, multiple joins, subject to the same disadvantages as those occurring in the above-described planarization process, are required in flow cells including a plurality of such fused elements. Further, the demands of machining the apexes from multiple elements, assembling the elements together, and joining them to attain repeatable polygonal conduits having the desired conduit dimensions make this approach both more costly and even less attractive as a production process. This approach becomes increasingly disadvantageous as the number of prismatic faces increases.
In characterizing various types of formed bodies by cytometric techniques, a recurring need is to simultaneously acquire several different types of optical signals resulting from interaction of the formed bodies with one or more radiation sources, i.e., some selected combination of multiple-wavelength fluorescence (F) signals, absorption (A) signals, and scatter (S) signals such as forward-scatter (FS), side-scatter (SS), or back-scatter (BS) signals; in such applications, the four external faces on a square/square prismatic flow cell requires that a plurality of sensors view an interrogation zone through complex beam-splitting and/or wavelength differentiating optics which, in addition to adding cost, introduce alignment and other optical difficulties. One might address this problem by adding more faces to the prismatic envelope and volumeter conduit of the flow cell, whereby both the flow cell envelope and internal conduit might have a pentagonal, hexagonal, heptagonal, etc., transverse cross-section, so that each optical measurement of interest could be made through a separate face of the flow cell. However, the manufacture of such prismatic flow cells by any of the above-noted conventional techniques would be so complex and time-consuming as to be totally impractical, especially if such flow cells were to be made in relatively large numbers for incorporation into commercial cytometric instruments.
It is known in the glass-working art to draw tubing from a larger preform of various inner and outer diameters between which is a cylindrical wall up to several centimeters in thickness. During the drawing operation, the preform is heated to a predetermined temperature at which its viscosity permits deformation, whereupon it is drawn axially, usually in a vertically downward direction, at a constant and predetermined rate. During this process, the diameters of the inner and outer cross-sections of the preform are substantially reduced with the original circular shapes being substantially retained, such shapes being the minimum energy shape, and the wall of the preform is significantly reduced in thickness. This drawing process has been adapted to form thick-wall transparent SiO2 tubing for use in producing single-piece (monolithic) flow cells, e.g., those used in the Coulter® Model LH750 automated hematology instruments as described in commonly assigned U.S. Pat. No. 5,125,737 (hereinafter the '737 patent) and manufactured and sold by Beckman Coulter, Inc. After a preform is drawn to a preferred inner diameter (e.g., 50 micra), the tubing is cut to a preferred length (e.g., about 6 mm), and a suitable flat (e.g., 1 mm wide) is lapped on the outer cylindrical surface of the tube which, as a result of the drawing has been reduced to a diameter of about 3.5 mm. The flat provides an optical port through which a radiation beam (e.g., from a HeNe laser) can be coupled, along a diameter, to the central cylindrical channel in the drawn tube. The flat is made substantially parallel to the channel axis at an arbitrary location on the outer cylindrical surface, e.g., to avoid an objectionable optical defect in the wall. The cylindrical channel is partially enlarged by boring from both ends to a suitable diameter (e.g., 1.25 mm), leaving in situ a short length (e.g., 65 micra) of the original channel in the middle of the flow cell that opens at each end into a small cup-shaped recess (e.g., of radius 600 micra) substantially tangent to a 90-degree cone coaxial with the original channel and continuous with the end bores. In use, the length of the original channel in the longitudinal section thus formed functions as a Coulter volumeter conduit, and the cylindrical bores communicate with external electrode chambers in a supporting structure, whereby the sample liquid in a hydrodynamically-focusing sheath liquid can be made to pass centrally through the volumeter conduit. Coulter V and C signals are acquired from the electrodes as formed bodies pass through the volumeter conduit; meanwhile, forward-scatter (FS) signals from the radiation beam are simultaneously acquired from the individual formed bodies as they pass through the cylindrical volumeter conduit.
Optical flow cells of the above type, i.e., truly monolithic flow cells made from a joinless, single piece of transparent material, inherently avoid many of the limitations and disadvantages described above for composite flow cells comprising joined transparent components of complementary geometries. Forward-scatter (FS) signals acquired from such flow cells permit reliable differentiation and enumeration of individual formed bodies when suitably correlated with Coulter V and C signals. However, because both the optically transparent envelope and the central interior volumeter conduit of such flow cells exhibit a substantially circular transverse cross-section, the wall of such flow cells acts as a non-axisymmetric lens, and scatter (S) signals acquired through it incur substantial optical aberrations that limit the ability to differentiate between certain types of formed bodies. While such differentiation can be improved by adding fluorescence (F) signals at different wavelengths, e.g., by selectively tagging the formed bodies with fluorescent dyes or dye-bead conjugates, such F signals are even more disadvantageously affected by optical aberrations than are the various scatter signals. Consequently, extensive effort has been directed toward flow cells having a prismatic channel surrounded by planar walls such as in the square/square geometry disclosed in the above '652 patent or for the triangular volumeter conduit disclosed U.S. Patent Application 2007/0085997 and its precursors, whereby such optical aberrations may be reduced. As noted, however, such flow cells are both difficult to produce using conventional production methods and subject to failure over time. Thus, although others have proposed flow cells within which such simultaneous optical, or optical and Coulter, measurements may be made on individual formed bodies flowing through a prismatic channel, no one has either reduced to practice a truly monolithic (i.e., joinless single-piece) flow cell suited to such applications or provided a technically enabling disclosure for doing so.